Process of preparing vitamin b12-active product from sewage sludge



Patented July 21, 1953 PROCESS 'OF PREPARING VITAMIN Biz-AC- V TIVEPRODUCT FROM SEWAGE SLUDGE ..Carl Shelley vMiner, Jr., Winnetka, andBernard Wolnak, Chicago, 111., assignors to Sewerage Commission of theCity ofMilwaukea Milwaukee, Wis a corporation of Wisconsin s No Drawing.ApplicationlMarch 3,1951,

1 tSeriaLNo. 213,794

invention relates to a product having high vitamin B12 activity and to aprocess-of {preparing the product and .haslfor anzobjec't the pro- Lclaims. (01. rev-s1 I vision of a product particularly suitable forincorporation into animal feeds. This application is acontinuation-in-part of our prior application Serial-No. 132,404, filedDecemberIIO, 1949. I,

At thepresent time there is a large commer cial "market for preparedfeeds for poultry and other nonruminant animals such as hogs. Such feedsas now commerciallysold compriseessentially proteins and carbohydratesofvegetable origin, such as are contained in meals obtained from Wheat,corn, rice, oats,.bar1ey,;1inseed, soybeans and cotton seed, :etc.Usually a prepared feed contains anombina'tionof mealsxof thischar acterin varying proportions. In order to provide alsatisfactory feed itiiasalso ibeen'the practice toincorporate various vitamins and mineralconstituents. Also itihas long zbeen beli'evedithat the vegetableprotein's containediin meals of this type must be :supplemented at:least to some extent with animal proteins, such as .'fish;meal or meatscraps; in order that. the animal fed may em- .ciently utilize all of"the vegetable proteins contained'within the feed..Generallyspeaking,:such.

animal proteins have been incorporated into feeds of .this character invarious amounts between about 2 per cent and about 5 per cent :in orderthat-the desired response may be obtained in the,

animals raised on the feed. -However, fish meal, meat scraps and otheranimal proteins representa substantial item of cost inlthepreparation offeeds of this general character and are availe ableonlyfin limitedamountsl Accordingly. .vari-' ous attempts-have been made in the past tocut down on the amount of, and to find cheap substi-l tutes for, theanimalprotei-ns .necessary to be added to feeds without adverselyaffecting. the .desired response of. the animal fed, It is now knownthatthis result-can be achieved by mixin with the; feed a growth factorwhichcontains .a thigh Y vitamin Biz activityn Vitamin B12 has come tobe recognized as'thegrowth factor or animal pro? tein factor (APE)which'issupplied tofe'eds :by the addition of meat "scraps, fish mealsand-the like. Y I 7 Accordingly it is one obj ectof this inve'n tion toprovide a productghavinga :high vitaniin-Bm activity which 'is suitablefor use vanimalfeeds and for other purposes A further object 'of thisinventiomisffthe pro vision of avitamin B12 active jn'iateriail"obtainable froinareadily'availablesource;f

Astillfurtherobjectoltliis meas re-ea provision of a product that maybesubstituted Wholly or in part for animal proteins normally considereddesirable in feeds for poultry and other nonruminant animals.

An additional object of this invention is the provision of a process ofpreparing a-concentrate suitable for use in the preparation of feeds.

An additional object of this invention is the provision 'of'-a proces'sfor preparing a producthaving a high vitamin B12 potency from a readilyavailable source material.

Y'Further 'anda'dditionalob' jectswill appear from r the followingdescription and the accompanying claims. I V

In accordance with-one embodiment'of this inverition a product having ahigh v'i-tamin'Biiactivity is one which comprises a water-solublefraction of a sludge resulting from the *microbiological treatment ofsewage." This aterso'luble fraction maybe obtained by leaching a driedsludge of this character with a suitable solvent such as water toextract the vitamin B12 active material, separating the undissolvedresidue "and then further concentrating the resulting solution byevaporation to form a residue or concentrate that has an extremely highvitamin B12 potency. ,4

7 Ina ccordance with a preferred embodiment-of this invention, thesludge employed for preparing the -concentrate is of the type known asactivated sludge. Dried activated sludge is now available commerciallyand sold under the trade name Milorganite. It is the product obtainedfrom that type of, "municipal sewage treatment and purification known inthe art as the activated sludge processi j "The commercial procedure for,obt'aininga dried form of activated sludge is somewhat as'fol-lo'ws:Raw sewage, sanitary and industrial, is brought to a sewage treatingplant by an-extensive system of municipal sewers. The incoming rawsewage is passed through partial sedimentation tanks and screens toremove large particles of suspended mineral and organic matter.

for-'exampl'aabout 25 per cent by volume of un- Thescreenedsewage isthen mixed with,

dried, freshly produced activated sludge which latter serves as aninoculant. The resulting mixture, termed m'ixed -l-iquor,. is thenpassed through large aeration tanks where it is agitated and aerated 'for four to six hoursfby a continuous stream of tiny air bubbles. The-airbubbles are formed in the mixed liquor by passing washed and compressedair through porous plates in the bottom of'tlie aeration tanks. Duringaeration theiinely organic matter the mixed sludge (containing'about 2%by weight of solids) is used to inoculate fresh incoming screened sewageas above described and the remaining sludge is treated with ferricchloride and filtered. The

ferric chloride serves to speed the filtering operation. The filter cakecontaining from. about 80% to about 85% moisture is dried to about 5%moisture with artificial heat in large revolving substance is driers.The resulting granular screened to uniform particle size, bagged andsold 5 such coagulants may be omitted entirely. By

the term activated sludge as used in thisspecification and in theappended claims is meant any substance which comprises essentially amass of microorganisms that is formed by the aeration of inoculatedsewage under conditions favoring microbial growth.

In accordance with this invention, the heatdried activated sludge may beextracted with water or other suitable solvent in which the vitamin B12active material is soluble and thereafter the undissolved solids areseparated from the resulting extract. The extract is then concentrated,as by evaporation, to remove at least a portion of the solvent whereby aconcentrate having a high vitamin B12 activity is formed. If desired theconcentrate may be further treated to further purify the vitamin B12potent fraction by known methods such as adsorption oncharcoal,'fractional extraction and crystallization and the like.

As disclosed in our prior application Serial No. 132,404, theconcentrate of this invention can be incorporated into any type ofpoultry or animal feed which contains substantial amounts of vegetableproteins and carbohydrates in order to replace in whole or in part theanimal proteins that have heretofore been considered necessary. Forexample, such feeds may. include singly or in various combinationssoybean meal, alfalfa leaf meal, linseed meal, cottonseed meal andvarious grain meals, such as those derived from wheat, corn, rice oats,buckwheat, barley,

4 Example In accordance with one embodiment of this invention, threeparts by weight of a mass of particulate heat-dried commercialMilorganite (80 mesh) wereadded to ten parts by weight of tap waterheated to C. The mixture was stirredand the temperature raised to aboutC., the hydrogen ion concentration being adjusted from a normally acidcondition to a value corresponding to pH 7010.5 by the occasionaladdition of 1 to 1 aqueous ammonia. After cooling overnight, the mixturewas reheated for five hours, filtered with suction and the filtrateevaporated on a steam bath to a heavy syrup. The syrup was thenevaporated to dryness in an oven at 70" C.-resulting in a brown solidwhich was easily ground down to a brown powder. This concentrate isuseful for the formulation of the feed described in our copendingapplication previously referred to.

In the preparation of a concentrate from Milorganite or dried activatedsludge in the manner indicated in the foregoing example, the temperatureof the extraction or leaching process may be above or below roomtemperature. Temperatures of 30 to 40 C. have been found to besatisfactory but higher temperatures up to the boiling point of thesolvent or higher usually result in more rapid extraction of the desiredwater-soluble fraction. It is convenient to operate at hydrogen ionconcentrations in the neighborhood of neutrality, however, since thevitamin Blz active factor is stable within the range of between about pH2 and about pH 12, it is permissibl'e to leach. and concentrate withinthis range. -It is preferred to employ particles of dried sludge whichare larger than those which will pass a 200 mesh screen inorder to avoiddifficulties in separating the undissolved sludge from the solventextract by filtration, centrifuging or otherwise. Other processingdetails also may be varied within wide limits, such as those relating totime of extraction, etc. In commercial operations. a percolation processis feasible in which the leaching solvent such as water is passedthrough one or more successive masses of the sludge whereby a highlyactive extract is obtainable which may then be concentrated byevaporation or otherwise.

Vitamin B12 assays have been made on solid concentrates prepared in themanner indicated in the foregoing specific example in accordance withvthe procedure disclosed by Peeler et al., Proc. Soc. Expl. Biol. andMed, "72, 5 15 (1949). These'assays showed an activity within the rangeof about 25 and about micrograms of vitamin B12 activity per gram ofsolid concentrate. Thus the materialprovides an excellent sourcematerial-'for'preparing an even more highly active vitamin B12 substanceby well known methods. For example, an aqueous solution of a concentratewas subjected to a charcoal adsorption procedure to remove the vitaminB12 active factor and the charcoal was leached with ethyl alcohol toextract the factor. The residue upon evaporation of the alcohol has ahigh potency which may be within the range of to 350 micrograms per gramof solids.

As indicated above, activated sludge is the preferred source of theconcentrate prepared in accordance with this invention. However, in itsbroaderaspects, this invention contemplates the use 'of other types .ofsludges resulting from the microbiological treatment of sewage. Forexample, digested activated sludge or sedimented digested sludge may beemployed. Digested activated sludge is theproduct formed by aerobicallytreating screenedgraw municipal sewage, passing the thus treatedmaterial to a settling tank, allowing the solids to settle, drawing off7 the supernatant liquid, passing the settled solids to a closed vessel,and digestingthe solids under anaerobic conditions. The solid materialwhich remains after the anaerobic digestion is separated and may bedried. Sedimented digested sewage sludge is prepared by passing screenedraw municipal sewage to settling tanks and withdrawing the settled solidmaterial to closed tanks wherein it is allowed to undergo an anaerobicdigestion. The solid material remaining after th anaerobicdigestion isseparated and dried.

Water extraction has been specifically disclosed in the foregoing forthe preparation of the concentrates, but it is clear that other solventscapable of leaching the water-soluble vitamin B12 factor from thesludge, such as isopropyl alcohol, ethyl alcohol, methyl alcohol,water-saturated butyl alcohol, water-diluted acetone, etc., may beemployed.

While a dried or heat-dried sludge derived fromthe microbiologicaltreatment of sewage is the preferred substance from which theconcentrate of this invention is prepared, it is also within the scopeof this invention to prepare it by the solvent extraction of the rawundried sludge. In such cases it may be preferableto subject. the rawundried sludge to an autolyzing step or to enzymatic digestion in orderto liberate the vitamin B12 .active factor, thus making it available forsolvent leaching or water extraction in the manner hereinbeforeindicated. An important aspect of this invention resides. in thetreatment While particular embodiments of this inven-.

tion are shown above, it will be understood, of

crobiological'treatment of sewage with water to leach out awater-soluble fraction thereof, separating undissolved solid sludge fromthe resulting aqueous solution, and concentrating said water-solublefraction in said solution whereby to produce said pro duct.

3. A process of preparing a product having a high vitamin B12 potencywhich comprises treating a heat-dried sludge resulting from themicrobiological treatment of municipal sewage with water to leach out awater-soluble fraction thereof, separating undissolved solid sludge fromthe resulting solution, and evaporating at least a portion of the Waterfrom said solution whereby to produce a concentrate having a highvitamin B12 potency.

4. A process of preparing a product having a high vitamin B12 potencywhich comprises leaching a mass of particles of heat-dried activatedsludgewith water at between about pH 2 and about pH 12 in order toextract a watersoluble fraction, said'particles initially being of asize greater than that which will pass through a -0 mesh screen,separating the resulting aqueous solution from undissolved solid sludge,and evaporating at least a portion of the water .from said solutionwhereby to produce a concentrate having a high vitamin B12 potency.

5. 'A process of preparing a concentrate containing an animal growthfactor having vitamin B12 activity which comprises contacting Iproduced.

course, that the invention is not to be limited thereto, since manymodifications may be made, and it is contemplated, therefore, by theappended claims, to cover any such modifications as fall within the truespirit and scope of this invention.

We claim:

1. A process of preparing a product having a high vitamin B12 potencyWhich comprises extractin 'with a solvent for vitamin B12 a watersolublefraction from a solid sludge resulting from the microbiologicaltreatment of sewage comprising a mass of microbial cells havingdisorganized cell walls whereby to produce a solu-'- tion having saidwater-soluble fraction dissolved therein, separating undissolved solidsludge from said solution, and concentrating said fraction in saidsolution whereby to produce said product. 2. A process of preparing aproduct having a high vitamin B12 potency which comprises treating adriedsolid sludge resulting from the mi- CARL SHELLEY MINER, JR.

BERNARD WOLNAK.

References Cited in the file of this patent UNITED STATES PATENTS NameDate Genter Apr. 30, 1935 Number Rickes Aug. 7, 1951 DeRose Aug. 28,1951 Meyer Apr, 29, 1952 OTHER REFERENCES pages 396 to 397.

Wolf Nov. 21, 1950

1. A PROCESS OF PREPARING A PRODUCT HAVING A HIGH VITAMIN B12 POTENCYWHICH COMPRISES EXTRACTING WITH A SOLVENT FOR VITAMIN B12 A WATERSOLUBLEFRACTION FROM A SOLID SLUDGE RESULTING FROM THE MICROBIOLOGICALTREATMENT OF SEWAGE COMPRISING A MASS OF MICROBIAL CELLS HAVINGDISORGANIZED CELL WALLS WHEREBY TO PRODUCE A SOLUTION HAVING SAIDWATER-SOLUBLE FRACTION DISSOLVED THEREIN, SEPARATING UNDISSOLVED SOLIDSLUDGE FROM SAID SOLUTION, AND CONCENTRATING SAID FRACTION IN SAIDSOLUTION WHEREBY TO PRODUCE SAID PRODUCT.